Toward Ratiometric Detection of NAD(P)H Quinone Oxidoreductase-1: Benzocoumarin-Based Fluorescent Probes

[Display omitted] •A rational approach of mismatched orbital energies to develop ratiometric probes in case they contain a fluorescence quenching substrate such as a quinone.•One of two new probes responded fast (10 min) to NQO1 with ratiometric signal changes.•Both the probes enabled fluorescence imaging of NQO1 levels in cells by two-photon microscopy. NAD(P)H quinone oxidoreductase-1 (NQO1) plays a role in reducing free radicals in cells and in detoxification of xenobiotics. It is also associated with some cancers and chemoresistance. Therefore, noninvasive tools for studying the enzyme are in demand, leading to about 30 activatable fluorescent probes containing a quinone substrate so far. However, most of them detect the enzyme with turn-on signaling, as the quinone substrate is apt to quench fluorescence through photo-induced electron transfer. We disclose a rational approach to develop a ratiometric NQO1 probe, which explores fluorophores with mismatched orbital energies for the fluorescence quenching. Two NQO1 probes based on an in cellulo super-bright and a highly water-soluble, deep-red emitting benzocoumarin fluorophores were synthesized and evaluated: They detected the enzyme, affording bright turn-on and good ratiometric images, respectively, as observed by one-photon as well as two-photon microscopic imaging. From molecular modeling and kinetics studies, not the enzymatic reduction but the subsequent chemical conversion seems to govern the response time. The result validates our approach to develop a ratiometric fluorescent probe in case it contains a fluorescence-quenching substrate. Additionally, the two probes display environment-insensitive emission behavior and thus hold promise for studying the enzyme in biological systems, which are heterogeneous in nature.